Off-line solid phase extraction and liquid chromatography-tandem mass spectrometry method for the quantitation of brivaracetam acid metabolites: Method validation and application to in vitro metabolism assays.

Brivaracetam (BRV) is a new high affinity synaptic vesicle protein 2A ligand recently approved for adults with partial-onset seizures. As a support to in vitro metabolism assays, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method coupled to off-line solid phase extraction (SPE) was developed to quantify BRV acid metabolites, that is, BRV-AC (carboxylic derivative derived from BRV hydrolysis) and BRV-OHAC (corresponding to hydroxylated BRV-AC). The method was validated for various incubates (liver and kidney tissue homogenates and blood, all from humans) and applied to in vitro metabolism assays. The analytes were isolated from buffered samples using ISOLUTE C8 96-well SPE plates. Chromatographic separation was achieved on a Waters Atlantis T3 C18 analytical column (2.1 mm × 50 mm, 5 µm) with detection accomplished using a Waters Premier tandem mass spectrometer in positive ion electrospray and multiple reaction monitoring (MRM) mode. The standard curves, which ranged from 1.00 to 200 ng/mL for BRV-AC, BRV-OHAC, were fitted to a 1/x2 weighted linear regression model. The intra-assay precision and inter-assay precision (expressed as coefficient of variation -%CV) were <8.5%, and the assay accuracy (deviation - %Dev) was within ±7.1% for the different matrices. This accurate, precise, and selective SPE/LC-MS/MS method has been successfully applied to in vitro assays aimed at characterizing the kinetics of BRV hydrolysis. BRV was found to be a better substrate for hydrolysis than its hydroxylated metabolite BRV-OH. BRV hydrolysis was detected in blood, liver and kidneys, demonstrating the broad distribution of the enzyme catalyzing the reaction.

Partial within-animal calibration: a new calibration approach in lead optimisation high-throughput bioanalysis.

The lead optimization phase of drug discovery requires high-throughput analyses for quantification in biological matrices and in plasma in particular. Over the last decade, some technical innovations allowed the pharmaceutical industry to improve the quality of the results. However, there is room for improvement. In this context, a new calibration strategy is proposed in this paper. Experiments were performed on dog plasma samples and it was showed that a within-animal calibration strategy can reduce the bias up to 20% and improve the precision up to 20%. However, a partial within-animal calibration is preferred to the full approach in order to avoid to many sample preparations.